Comparison of biomarker and chromatographic analytical approaches to pharmacokinetic study of sitagliptin.

The pharmacokinetic profiling of active compounds is necessary for drug development and application. Approaches to a pharmacokinetic study based on biological markers are alternatives to traditional approaches based on chromatographic methods. The aim of the study was to compare two analytical approaches to pharmacokinetics investigation for an example of sitagliptin in rabbits after one dose oral administration. The method for sitagliptin quantification in rabbit plasma samples based on a correlation between its concentration and dipeptidyl peptidase IV activity was proposed, validated, and applied. The high-performance liquid chromatography (HPLC)-ultraviolet (UV) method was also validated and applied for the same sample analysis. The plasma pharmacokinetics of sitagliptin after oral administration to the rabbits in one dose was characterized after two analytical assays. The close values of the main pharmacokinetic parameters were obtained after two approaches. The nontraditional approach based on correlation of special marker activity and active substance concentration appears to be more sensitive than HPLC-UV. Thus, the sitagliptin concentrations determined by biomarker assay were higher than the lower limit of quantification (LLOQ) for a longer period (more timepoints) than after the HPLC-UV assay. This feature may influence the values of some calculated concentration-dependent (area under the curve [AUC]0-t , etc.) and time-dependent parameters (mean residence time [MRT], T1/2 , etc.). The values of Tmax obtained by the two approaches were similar and adequate for oral drug administration that confirms the correctness of biomarker selection for pharmacokinetics assessment. The obtained results on the example of sitagliptin confirms that the biomarker approach is adequate and applicable for a pharmacokinetics study. Similar approaches may be effective for individual compounds and complex mixtures when it is difficult or impossible to analyze them traditionally by chromatographic methods.

Variability of Major Phenyletanes and Phenylpropanoids in 16-Year-Old Rhodiola rosea L. Clones in Norway.

Rhodiola rosea L. (roseroot) is an adaptogen plant belonging to the Crassulaceae family. The broad spectrum of biological activity of R. rosea is attributed to its major phenyletanes and phenylpropanoids: rosavin, salidroside, rosin, cinnamyl alcohol, and tyrosol. In this study, we compared the content of phenyletanes and phenylpropanoids in rhizomes of R. rosea from the Norwegian germplasm collection collected in 2004 and in 2017. In general, the content of these bioactive compounds in 2017 was significantly higher than that observed in 2004. The freeze-drying method increased the concentration of all phenyletanes and phenylpropanoids in rhizomes compared with conventional drying at 70 °C. As far as we know, the content of salidroside (51.0 mg g-1) observed in this study is the highest ever detected in Rhodiola spp. Long-term vegetative propagation and high genetic diversity of R. rosea together with the freeze-drying method may have led to the high content of the bioactive compounds observed in the current study.

Natural Deep Eutectic Solvents for the Extraction of Phenyletanes and Phenylpropanoids of Rhodiola rosea L.

The extraction of Rhodiola rosea rhizomes using natural deep eutectic solvent (NADES) consisting of lactic acid, glucose, fructose, and water was investigated. A two-level Plackett-Burman design with five variables, followed by the steepest ascent method, was undertaken to determine the optimal extraction conditions. Among the five parameters tested, particle size, extraction modulus, and water content were found to have the highest impact on the extrability of phenyletanes and phenylpropanoids. The concentration of active compounds was analyzed by HPLC. The predicted results showed that the extraction yield of the total phenyletanes and phenylpropanoids (25.62 mg/g) could be obtained under the following conditions: extraction time of 154 min, extraction temperature of 22 °C, extraction modulus of 40, molar water content of 5:1:11 (L-lactic acid:fructose:water, mol/mol), and a particle size of rhizomes of 0.5-1 mm. These predicted values were further verified by validation experiments in predicted conditions. The experimental yields of salidroside, tyrosol, rosavin, rosin, cinnamyl alcohol and total markers (sum of phenyletanes and phenylpropanoids in mg/g) were 11.90 ± 0.02, 0.36 ± 0.02, 12.23 ± 0.21, 1.41 ± 0.01, 0.20 ± 0.01, and 26.10 ± 0.27 mg/g, respectively, which corresponded well with the predicted values from the models.

Pharmacokinetic Study of Bioactive Glycopeptide from Strongylocentrotus droebachiensis After Intranasal Administration to Rats Using Biomarker Approach.

A glycopeptide fraction (GPF) from internal organs of green sea urchins (Strongylocentrotus droebachiensis Müller, Strongylocentrotidae) has been reported to be an effective bronchitis treatment. In this study, we evaluated the pharmacokinetic and tissue distribution of GPF, following single and repeated intranasal (i/n) administration over the course of seven days in rats. The method measuring lactate dehydrogenase as biomarker was used to analyse the plasma and tissue concentrations of GPF. GPF appears in the plasma 15 min after single i/n administration (100 µg/kg) and reaches its maximum at 45 min. The area under the curve (AUC)0-24 and Cmax were similar using both i/n and intravenous administration, while mean residence time (MRT) and T1/2 after i/n administration were significantly higher compared with intravenous (i/v) administration. The absolute bioavailability of GPF after i/n administration was 89%. The values of tissue availability (ft) provided evidence about the highest concentration of GPF in the nose mucosa (ft = 34.9), followed by spleen (ft = 4.1), adrenal glands (ft = 3.8), striated muscle (ft = 1.8), kidneys (ft = 0.5), and liver (ft = 0.3). After repeated dose administration, GPF exhibited significantly higher AUC0-24 and MRT, indicating its accumulation in the plasma.

Pharmacokinetic and Tissue Distribution of Fucoidan from Fucus vesiculosus after Oral Administration to Rats.

Fucus vesiculosus L., known as bladderwrack, belongs to the brown seaweeds, which are widely distributed throughout northern Russia, Atlantic shores of Europe, the Baltic Sea, Greenland, the Azores, the Canary Islands, and shores of the Pacific Ocean. Fucoidan is a major fucose-rich sulfated polysaccharide found in Fucus (F.) vesiculosus. The pharmacokinetic profiling of active compounds is essential for drug development and approval. The aim of the study was to evaluate the pharmacokinetics and tissue distribution of fucoidan in rats after a single-dose oral administration. Fucoidan was isolated from F. vesiculosus. The method of measuring anti-activated factor X (anti-Xa) activity by amidolytic assay was used to analyze the plasma and tissue concentrations of fucoidan. The tissue distribution of fucoidan after intragastric administration to the rats was characterized, and it exhibited considerable heterogeneity. Fucoidan preferentially accumulates in the kidneys (AUC0–t = 10.74 µg·h/g; Cmax = 1.23 µg/g after 5 h), spleen (AUC0–t = 6.89 µg·h/g; Cmax = 0.78 µg/g after 3 h), and liver (AUC0–t = 3.26 µg·h/g; Cmax = 0.53 µg/g after 2 h) and shows a relatively long absorption time and extended circulation in the blood, with a mean residence time (MRT) = 6.79 h. The outcome of this study provides additional scientific data for traditional use of fucoidan-containing plants and offers tangible support for the continued development of new effective pharmaceuticals using fucoidan.

Pharmacokinetics and Tissue Disposition of Nanosystem-Entrapped Betulin After Endotracheal Administration to Rats.

BACKGROUND AND OBJECTIVES: Betulin is a triterpene extracted from the cork layer of the outer bark of Betula spp. It has a wide spectrum of pharmacological activities, including being lung protective; however, its bioavailability is low. To increase its bioavailability, betulin was entrapped in a nanosystem (BN). In this study, we investigated the pharmacokinetics and tissue distribution of nanosystem-entrapped betulin after single dose endotracheal administration to rats. METHOD: Betulin was nanosystem-entrapped using a solvent exchange technique. The surface morphology and size of the nanosystem were characterized by transmission electron microscopy and dynamic light scattering. The plasma and tissue concentrations of betulin were determined using a validated high-performance liquid chromatography method. RESULTS: The highest concentration of betulin was found in lungs and liver, and the lowest in the heart. Betulin did not penetrate highly vascularized tissues or tissue with an average degree of vascularization, nor did it cross the blood-brain barrier. Tissue availability in the lungs was 1.3 times higher for BN than for free betulin. Betulin was detected in the bloodstream at 15 min after administration of BN compared with only at 1 h after administration of free betulin. Penetration of betulin in the liver tissue was characterized by a high degree of intensity both for BN and free betulin. Betulin in the heart tissue was detected in much smaller quantities than in the liver. CONCLUSION: Entrapment of betulin in nanosystem form shows promise as a novel strategy in the treatment of pulmonary diseases.

Antiallergic effects of pigments isolated from green sea urchin (Strongylocentrotus droebachiensis) shells.

This study was undertaken to evaluate possible antiallergic effects of an extract of pigments from green sea urchin (Strongylocentrotus droebachiensis) shells. Effects were studied on animal models - guinea pig ileum contraction, rabbit eyes allergic conjunctivitis, and rabbit local skin irritation. The extract significantly reduced, in a dose-dependent manner, the histamine-induced contractions of the isolated guinea pig ileum with ID50 =1.2 µg/mL (in equivalents of spinochrome B), had an inhibitory effect on the model of ocular allergic inflammation surpassing the reference drug olopatadine, and did not show any irritating effect in rabbits. The extract predominantly contained polyhydroxy-1,4-naphthoquinone which would be responsible for the pharmacological activity. The active compounds of the extract were evaluated in silico with molecular docking. Molecular docking into H1R receptor structures obtained from molecular dynamic simulations showed that all spinochrome derivatives bind to the receptor active site, but spinochrome monomers fit better to it. The results of the present study suggest possibilities for the development of new agents for treating allergic diseases on the base of pigments from sea urchins shells.

Birch bark extract as therapy for chronic hepatitis C--a pilot study.

The hepatoprotective effect of birch bark extract (BBE) in patients with chronic hepatitis C (CHC) was studied. Forty-two patients with serologically confirmed chronic hepatitis C were treated for 12 weeks with 160 mg standardized BBE per day. The primary outcome parameter measured was the rate of alanine aminotransferase (ALT) normalization after 12 weeks. Secondary parameters included the course of ALT, aspartate aminotransferase (AST) levels, quantitative HCV RNA levels, subjective symptoms associated with CHC (fatigue, abdominal discomfort, depression, and dyspepsia), safety and compliance. The qualitative-quantitative analysis of BBE was made using high performance liquid chromatography to confirm the presence of 75% betulin and 3.5% betulinic acid. Significant differences in the mean ALT and HCV RNA levels were observed after 12 weeks of treatment. The level of ALT was decreased in 54.0% and normalized (p=0.046). HCV RNA was reduced in 43.2% (p=0.016). After 12 weeks of treatment, reports of fatigue and abdominal discomfort were reduced by 6-fold (p=0.028) and 3-fold (p=0.05), respectively. Dyspepsia was no longer reported (p=0.042) and the effect was significantly different from baseline. Because this study lacks a control group clinical relevance of the data can only be estimated in future by following controlled clinical trials.

Determination and pharmacokinetic study of taxifolin in rabbit plasma by high-performance liquid chromatography.

Taxifolin has been widely used in the treatment of cerebral infarction and sequelae, cerebral thrombus, coronary heart disease and angina pectoris. A reliable sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV detection for the pharmacokinetic study of taxifolin in rabbit plasma after enzymatic hydrolysis was developed and validated for the first time. Taxifolin, with biochanin A as the internal standard, was extracted from plasma samples by liquid/liquid extraction after hydrolysis with beta-glucuronidase and sulfatase. Chromatographic separation was conducted on a Luna C18 column (4.6 mm x 150 mm, 5 microm particle size) and pre-column (2.0 mm, the same sorbent). Two-step linear gradient elution with acetonitrile and 0.03% water solution of trifluoroacetic acid as mobile phase at a flow rate of 1.0 ml/min was used. The UV detector is set at 290 nm. The elution time for taxifolin and biochanin A was approximately 7.9 and 18.3 min, respectively. The calibration curve of taxifolin was linear (r > 0.9997) over the range of 0.03-5.0 microg/ml in rabbit plasma. The limit of detection (LOD) and limit of quantification (LOQ) for taxifolin were 0.03 and 0.11 microg/ml, respectively. The present method was successfully applied for the estimation of the pharmacokinetic parameters of taxifolin following intravenous and oral administration of lipid solution to rabbits. The absolute bioavailability of taxifolin after oral administration of lipid solution was 36%.

Determination of icariin in rat plasma by reverse-phase high-performance liquid chromatography after oral administration of a lipid-based suspension of Epimedium koreanum extract.

A simple, specific and sensitive method for quantitative determination of icariin in rat plasma using reverse-phase high-performance liquid chromatography with UV-detection was developed and applied to an animal study of a lipid-based suspension of the Epimedium koreanum extract in rats. Rutin was selected as the internal standard and methanol was found to be the best solvent for extraction of icariin from the plasma. Linearity was observed between 0.030 and 100 microg/mL (r > 0.99). The extraction recoveries of icariin and rutin were approximately 75 and 80%, respectively, in plasma. The intra- and inter-day coefficients of variation were less than 5%. The limit of detection was 6 ng/mL and the limit of quantification was 18 ng/mL.

Comparison of high performance TLC and HPLC for separation and quantification of chlorogenic acid in green coffee bean extracts.

Two chromatographic methods, high-performance TLC (HPTLC) and HPLC, were developed and used for separation and quantitative determination of chlorogenic acid in green coffee bean extracts. For HPTLC silica gel Kieselgel 60 F 254 plates with ethyl acetate/dichlormethane/formic acid/acetic acid/water (100:25:10:10:11, v/v/v/v/v) as mobile phase were used. Densitometric determination of chlorogenic acid by HPTLC was performed at 330 nm. A gradient RP HPLC method was carried out at 330 nm. All necessary validation tests for both methods were developed for their comparison. There were no statistically significant differences between HPLC and HPTLC for quantitative determination of chlorogenic acid according to the test of equality of the means.

Comparison between HPLC and HPTLC densitometry for the determination of icariin from Epimedium koreanum extracts.

Dry extracts of the aerial parts of Epimedium koreanum were quantified by HPLC and high performance TLC (HPTLC). A gradient HPLC method was used for the quantification of the prenylflavone glycoside icariin at 270 nm. A direct HPTLC assay was developed for the determination of icariin at 270 nm. The UV detection of both analytical assays were used to examine the purity of icariin peaks and compared with the standards. The assays provide good accuracy, reproducibility, and selectivity for the quantitative analysis of icariin. The icariin contents of five different dry extracts were compared by HPLC and HPTLC densitometry. The quantitative results of both analytical methods did not show any statistically significant differences between them, although a trend to slightly lower mean values could be found for the HPLC method.